One of the most pressing challenges to the biomedical research community lies in the development of novel antimicrobials as a means of combating the evolution of antibiotic-resistant pathogens. As such, research in the van der Donk is focused on the study of lanthipeptides, a class of ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products, which have demonstrated significant potential as antibiotics. Indeed, the antibiotic lanthipeptide, nisin, has been used in the U.S. food industry for almost half a century with very few reports of antibiotic resistance. These natural products derive their name from the presence of lanthionine rings within their structure, which are formed through the conjugation of cysteine thiols to dehydroalanine and dehydrobutyrine residues within the maturing lanthipeptide. It has recently been found that LanBs, the enzymes responsible for the generation of dehydroalanine and dehydrobutyrine residues in some lanthipeptides, rely upon glutamyl-tRNAGlu in order to glutamylate and subsequently eliminate the hydroxyl-group from serine and threonine. Unfortunately, little is known about the enzymatic mechanisms by which this occurs or how it is regulated. Therefore, through the use of isotopically labeled cofactors, non-reactive substrate mimics, mutagenesis, and enzyme kinetics analysis, the research proposed herein is designed to examine the chemical and structural features that underlie LanB activity. The specific goals to be achieved include the determination of the mechanism and chemoselectivity of glutamate transfer by NisB, the structural elucidation of the glutamylation and elimination active sites, and the molecular mechanisms that determine tRNA cofactor recognition. Through a better understanding of these properties, our overall objective is to better understand lanthipeptide biosynthesis in order to inform the development of novel antibiotic and therapeutic compounds.